We previously purified and characterized an arachidonoyl diacylglycerol kinase and showed that it is present in the testis, brain, and retina of baboons. The enzyme is one of at least ten diacylglycerol kinases that are present in mammalian tissues. We have now partially purified and characterized an additional diacylglycerol kinase, which is coexpressed with the arachidonoyl diacylglycerol kinase. The diacylglycerol kinase is soluble, but can bind to unilamellar lipid vesicles in vitro. Two types of binding sites seem to be involved, one that interacts with vesicle-associated acidic lipids and one that interacts with vesicle-associated diacylglycerol. Both types of binding sites must be engaged for effective enzyme-vesicle interaction. Under these conditions and when both Mg2+ and ATP are present in the medium, enzyme-vesicle interaction is optimal and correlates with catalysis. Even so, linear reaction rates are a complex function of what others have termed enzyme "scooting " and "hopping" phenomena. The enzyme apparently binds to the surface of a vesicle, catalyzes a few reactions, then dissociates from that surface, binds to a new vesicle, and begins a new round of catalysis. FUNDING NIH grant RR00166 and the Howard Hughes Medical Institute.